PROJECT SUMMARY/ABSTRACT Owing to new regulatory guidelines, the organic anion transporter (OAT1)--a transporter discovered by the PI's group (as NKT) and which is involved in the elimination of many common drugs--has received considerable attention. But nearly all the published studies continue to focus on common drugs rather than endogenous substrates of the transporter, which is highly conserved from an evolutionary standpoint. Metabolomics analysis of our Oat1 knockout mice (Oat1KO), as well as in vitro transport data, indicate that OAT1 is a major transporter of metabolites, signaling molecules, vitamins, gut microbiome products, and antioxidants. Our previous genome-scale metabolic reconstructions of altered metabolism of the Oat1KO mouse under normal dietary conditions enabled us to create the first multispecific drug transporter (OAT1)- centered metabolic network--which was further supported by considerable metabolomics and in vitro data. Many pathways (e.g., fatty acids, gut microbiome products, branched chain amino acids) in this largely validated network of ~150-200 metabolites are also known to be important in metabolic diseases such as diabetes, metabolic syndrome, obesity, and gut microbiome-associated illness. In this proposal: 1) We will use dietary modifications (e.g., high fat, branched chain amino acids) and antibiotic treatment of the Oat1 KO mice to define the role of Oat1 under conditions applicable to metabolic diseases. We will test the binding of metabolites revealed by metabolomics under the various dietary conditions (or disease models) using a high throughput unlabeled (BLI) assay to quantify the interaction of metabolites with OAT1; this information will be used to prioritize metabolites for subsequent uptake assays involving the use of radiolabeled compounds. 2) We will then use this information, together with gene expression data from the knockout tissues under various dietary conditions, to reconstruct a metabolic network for each of these dietary (pathological model) conditions- -using methods we have previously successfully used to create an OAT1-centered metabolic network under normal dietary conditions. Together, these studies will help define the unique roles of OAT1 in regulation of aberrant metabolism in these dysregulated metabolic states. Finally, we discuss how this information will set the stage for understanding aspects of drug-induced metabolic syndrome in patients taking OAT1- transported drugs. We have proven expertise in the necessary techniques and a team of world-class collaborators to ensure success.